The present invention relates to a filtration-detection device, which can be used for rapid hybridization assays. Methods of using the device employ marker-loaded particles, e.g., liposomes, and either electrochemical or optical detection of an analyte in a test sample.
Hybridization assays, in the form of enzyme-linked/radiolabel-linked nucleic acid probing (i.e. Southern and Northern blot analysis), have been widely used in the area of clinical diagnostic and research laboratory analysis for the detection of specific nucleic acid sequences. These methods offer high specificity, sensitivity, and ease of operation over other standard laboratory procedures. However, some of the disadvantages of the current probing technology which necessitate further improvement on the methodology include the lengthy time required for target-probe interaction, reagent additions, enzymatic conversion of substrate, and numerous washing steps between the various operations.
As an alternative to the use of enzymes or radioactivity, liposomes are of interest as detectable labels in hybridization assays because of their potential for immediate signal amplification. Liposomes are spherical vesicles in which an aqueous volume is enclosed by a bilayer membrane composed of phospholipid molecules (New, Liposomes: A Practical Approach, IRL Press, Oxford (1990)). Previous studies (Plant et al., Anal. Biochem., 176:420-426 (1989); Durst et al., In: GBF Monograph Series, Schmid, Ed., VCH, Weinheim, FRG, vol. 14, pp. 181-190 (1990)) have demonstrated the advantages of liposome-encapsulated dye over enzymatically produced color in the enhancement of signals in competitive immunoassays. The capillary migration or lateral flow assays utilized in these experiments, avoid separation and washing steps and long incubation times and attains sensitivity and specificity comparable to enzyme-linked detection assays. Nevertheless, the methodologies (Siebert et al., Anal. Chim. Acta, 282:297-305 (1993); Roberts et al., Anal. Chem., 67:482-491 (1995); Siebert et al., Anal. Chim. Acta, 311:309-318 (1995); Reeves et al., Trends Anal. Chem., 14:351-355 (1995); Rule et al., Clin. Chem., 42:1206-1209 (1996)) involve operations and solutions that make the handling of the sample and reagents susceptible to errors and more difficult to use for untrained personnel. Despite improvements in handling (U.S. Pat. No. 5,985,791 to Roberts et al.), lateral flow technologies have some limitations with regard to electrochemical detection.
The present invention is directed to overcoming the above-noted deficiencies in the prior art.
The present invention relates to a filtration-detection device for detecting or quantifying an analyte in a test sample. The filtration-detection device includes a filtration device having a first binding material immobilized thereto, wherein the first binding material is capable of binding to a portion of the analyte, and a detection assembly positioned relative to the filtration device to detect or quantify analyte bound to the first binding material.
The present invention also relates to a method for detecting or quantifying an analyte in a test sample. This method involves providing a filtration device having a first binding material immobilized thereto, wherein the first binding material is capable of binding to a portion of the analyte, providing a test mixture including the test sample and a binding material conjugate, wherein the binding material conjugate includes a second binding material bound to a first marker complex, the first marker complex includes a particle and a marker, and the second binding material is selected to bind with a portion of the analyte other than the portion of the analyte for which the first binding material is selected, passing the test mixture through the filtration device under conditions effective to permit reaction between any analyte present and the first and second binding materials, detecting the presence or amount of the marker on the filtration device using a detection assembly, and correlating the presence or amount of the marker on the filtration device with the presence or amount, respectively, of the analyte in the test sample.
The present invention further provides a method for detecting or quantifying an analyte in a test sample involving providing a filtration device having a first binding material immobilized thereto, wherein the first binding material is capable of binding to a portion of the analyte, providing a test mixture including the test sample and an analyte analog conjugate, wherein the analyte analog conjugate includes an analyte analog bound to a first marker complex, and the first marker complex includes a particle and a marker, passing the test mixture through the filtration device under conditions effective to permit competition to occur between any analyte present and the analyte analog conjugate for the first binding material, detecting the presence or amount of the marker on the filtration device using a detection assembly, and correlating the presence or amount of the marker on the filtration device with the presence or amount, respectively, of the analyte in the test sample.
The present invention also relates to a method for detecting or quantifying the amount of an analyte in a test sample which includes providing a test mixture including the test sample, a first conjugate including a first binding material bound to a first marker complex, wherein the first marker complex includes a particle and a marker, and wherein the first binding material is selected to bind with a portion of the analyte, and a second conjugate including a second binding material bound to a second marker complex, wherein the second marker complex includes a particle and a marker, and wherein the second binding material is selected to bind with a portion of the analyte other than the portion of the analyte for which the first binding material is selected, permitting reaction to occur in the test mixture between any analyte present and the first and second binding materials to form a first conjugate-analyte-second conjugate aggregate, collecting the aggregate on a filtration device, detecting the presence or amount of the marker on the filtration device using a detection device, and correlating the presence or amount of the marker on the filtration device with the presence or amount, respectively, of the analyte in the test sample.
The device and methods of the invention can be used directly in the field. The device can be used repeatedly or can be used only once. When used only once, the device can be free from residual environmental contaminants other than what may be present in the sample to be measured. Samples can be assayed within minutes after collection, with the results immediately available on-site. In addition, the device and methods of the invention are less complex than many of the prior materials and methods. The ability to deliver quantitative results without additional steps for spectrophotometric or fluorometric analysis is an advantage of the present electrochemical device and method over devices and methods that employ dyes and fluorescent materials as markers.
In addition, in one embodiment of the invention, electroactive marker-loaded liposomes as used in the device and method of the invention provide a highly sensitive, rapid, or even instantaneous signal production/amplification system. Furthermore, in some embodiments of the invention, the amount of marker measured on the filtration device of the test device is directly proportional to the analyte concentration in the sample. This feature of the invention provides a particular advantage over prior test devices, nucleic acid detection assays, and immunoassays, providing an intuitive correlation between signal strength and analyte concentration. Electrochemical detection offers greater sensitivity than colorimetric determination and is comparable to fluorometry or chemiluminescence. In addition, the present invention provides quantitative results that can be obtained directly from the electroanalyzer or other detection instrumentation to which the test device is connected, without the need to transfer the device to a separate optical measurement device. Also, electrochemical detection allows for testing in solutions or mixtures that are highly colored or include particulate matter, for which optical detection may be unsuitable.
Interdigitated electrode arrays are particularly suitable for the test device and methods of the present invention due to their planar configuration and their inherent sensitivity for electrochemical measurements. Microelectrodes fabricated in an interdigitated array have inherent advantages in signal detection over more conventional electrode configurations. These advantages can only be realized with electrodes of very small dimensions due to the theoretical relationships between electrode geometry and ionic diffusion. Scaling down the size of an individual electrode has the advantage of increasing the rate of mass transport, increasing the signal-to-noise (faradaic/charging current) ratio, and decreasing ohmic signal losses, as described in Fleischmann et al., Eds. Ultramicroelectrodes, Datatech Systems, Inc., Morganton, N.C. (1987), which is hereby incorporated by reference. Advantages of microelectrodes are also described in Howell, Voltammetric Microelectrodes, Bioanalytical Systems, Inc., West Lafayette, Ind. 47906, which is hereby incorporated by reference.
Advantages of fabricating small electrodes in interdigitated arrays go even further by allowing redox cycling of ions back and forth between anode(s) and cathode(s). See Niwa et al., Anal. Chem. 65:1559-1563 (1993) and Niwa et al., Anal. Chem. 66:285-289 (1994), each of which is hereby incorporated by reference. This generates much larger currents for detection and allows for the use of extremely small sample volumes. By using a dual potentiostat and a four-electrode system with an interdigitated array, it is possible to almost completely eliminate charging current. This results in a greater signal-to-noise ratio and allows for the use of extremely high scan rates. See Niwa et al., Anal. Chem. 62:447-452 (1990) and Chidsay et al., Anal. Chem. 58:601-607 (1986), which are hereby incorporated by reference. Furthermore, the sophisticated electronics needed to detect the very small currents associated with individual microelectrode filaments are not necessary due to the summation of current from the large array of microelectrodes.